WO2020257753A1 - Phosphatidylserine decarboxylase inhibitors - Google Patents

Abstract

Provided herein are high throughput screening methods for identifying inhibitors of phosphatidylserine decarboxylase. These inhibitors are useful as anti-bacterial and/or anti-fungal agents.

Description

TITLE OF THE INVENTION

Phosphatidylserine Decarboxylase Inhibitors

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 62/864,290 entitled "HIGH-THROUGHPUT SCREENING FOR PHOSPHATIDYLSERINE DECARBOXYLASE INHIBITORS USING A DISTYRYLBENZENE-BIS-ALDEHYDE (DSB-3)-BASED FLUORESCENCE ASSAY," filed June 20, 2019, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI123321, AI138139, AI136118 and AI097218 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Phosphatidylethanolamine (PE) is a zwitterionic phospholipid that plays an essential role in cellular functions, both as a major structural lipid of cellular membranes, as well as a substrate for multiple phospholipid-dependent reactions. In Gram negative prokaryotes like Escherichia coli , PE is the principal glycerophospholipid, whereas in eukaryotes, it is typically the second most abundant glycerophospholipid. In E. coli, PE accounts for -75% of total phospholipids, all of which is generated from phosphatidylserine (PS) by a PSD associated with the inner cytoplasmic membrane. The contribution of the latter pathways to the synthesis of PE varies among organisms and tissue types. For example, while the majority of PE produced in rat hepatocytes and hamster heart is generated by the CDP- ethanolamine pathway, in cultured Chinese hamster ovary cells and baby hamster kidney cells, more than 80% of PE is produced from the decarboxylation of PS, even in the presence of ethanolamine.

Because of their essential role in cell viability, PSD enzymes hold promise as targets for the development of new classes of antimicrobials and anti-tumor agents. However, such efforts have heretofore been hamstrung by challenges associated with large-scale production of these membrane-bound enzymes, and the lack of effective, robust, low-cost, automation- amenable enzyme assay for high throughput screening (HTS). Traditional methods for determining PSD activity in vitro have relied on the use of ³H-PS or ¹⁴C-PS as a substrate. ³H-PE or ¹⁴C-PE can be reliably analyzed by thin layer chromatography; however, the assay cannot be miniaturized to a format compatible with HTS. Alternatively, PSD activity can be determined following detection of released ¹⁴C0₂ from ¹⁴C- PS (19). This method, however, is technically incompatible with large-scale screening.

There is a need in the art for novel methods of screening a plurality of drug candidate compounds against a target phosphatidylserine decarboxylase (PSD) enzyme, as well as for identifying inhibitors of that enzyme. The present disclosure addresses these needs.

BRIEF DESCRIPTION OF THE FIGURES

The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

FIGs. 1A-1D show a 384-well high throughput screening campaign for biochemical inhibitors of Plasmodium knowlesi PSD. FIG. 1 A shows the PS to PE catalytic reaction is performed at 24 °C for 75 minutes. After pH shift to arrest catalysis, fluorescent adducts are generated by addition of 10 pM DSB-3 (4,4'-((lA, l'A)-(2,5-bis((2,5,8,12, 15,18- hexaoxanonadecan-10-yl)oxy)-l,4-phenylene)bis(ethene-2,l-diyl))dibenzaldehyde). The fluorescence in each well is quantified using l_6c = 403 nm and Z_em = 508 nm. FIG. IB shows a primary HTS Z’ plot. HTS was performed using 384-well plates, with each well containing 30 ng (-12.5 nM) Mbp-His6-A34PkPSD and 1 nanomole (50 mM) PS. Test compounds were administered at a 10 mM final concentration. FIG. 1C shows plate-wise tracking of signal -to- background from primary HTS assay plates. N=420, 384-well plates tested in 10 assay runs. FIG. ID shows plate-wise tracking of PSD inhibition with HTS assay plates. N=420, 384- well plates tested in 12 assay runs.

FIGs. 2A-2B show summary of n=3 single concentration retests. FIG. 2A shows triplicate 10 mM retests of 851 primary hits with parallel fluorescent interference counter assay (interference data not shown; briefly, all wells received phosphatidylethanolamine + DSB-3 to generate max emission, and compounds were evaluated for ability to trigger false positives by quenching fluorescent emission in the absence of an enzymatic reaction) yielded 96 confirmed, non-fluorescent hits. FIG. 2B shows reduction of the set of confirmed hits queued for concentration-response testing from fresh powder from 96 to 36.

FIGs. 3A-3B shows confirmation of primary hits from fresh powders by retesting. FIG. 3A shows 36 confirmed HTS hits were selected to progress to biochemical

concentration response studies from commercially procured dry powders to confirm the activity of the parent structure. 15/36 confirmed hits were available commercially as dry powders (filled circles). After the initial concentration-response study on these 15 compounds, 60 analogs were procured via custom synthesis or via virtual hit expansion (VHE) from the commercially available chemical space (open circles). FIG. 3B shows active primary hits were counter-screened for mammalian cytotoxicity in HeLa and HepG2 cells. Lead candidates were triaged versus potential toxicity to host cells via a 72 h CellTiter-Glo- based cytotoxicity readout in HeLa cells. Potential for liver-specific toxicity was evaluated by an identical readout in HepG2 cells. Compounds displaying a minimum 3-fold cytotoxicity margin versus one or both cell lines and negligible fluorescent assay interference progressed to cell-based anti-fungal assay testing. Despite limited initial potency, YU253467 was subsequently shown to exert ethanolamine-dependent anti-fungal activity, indicating a potential for PSD engagement in the anti-fungal mechanism.

FIGs. 4A and 4B show cell-based orthogonal assay for activity confirmation. Growth of C. albicans growth in the absence (vehicle) or presence of YFJ253467, YU253454, YU224252 and 196325 in medium lacking or supplemented with 2 mM ethanolamine (Etn) (FIGs. 4A and 4B). Fluconazole was included as a positive control for anti- fungal activity (FIG. 4B).

FIGs. 5A-5D show dose-dependent inhibition of C. albicans by YU253467 and its analog YU254403. FIG. 5 A shows the structure of YU254403. FIGs. 5B and 5C show the MIC₅₀ of YU253467 increased from 22.5 pg/mL in the absence of ethanolamine to 75 pg/mL in the presence of ethanolamine (FIG. 5B), whereas that of YU254403 increased from 15 pg/mL in the absence of ethanolamine to 60 pg/mL in the presence of ethanolamine (FIG. 5C). FIG. 5D shows fluconazole displayed an MIC₅₀ of 2.5 pg/mL and a characteristic insensitivity to ethanolamine supplementation.

FIGs. 6A-6B show efficacy of YU253467 and YU254403 against C. albicans lacking I* SI) / or PSD2 genes. FIG. 6A shows in vitro efficacy of YU253467 and its analog

YU254403 against C. albicans wild type and knockout strains lacking PSD1 or PSD2 activity. Growth assays were conducted in medium lacking or supplemented with 2 mM ethanolamine (Etn) in the absence or presence of the compound at 75 pg/mL or 200 pg/mL. FIG. 6B shows activity of YU253467 and YU254403 against recombinant PfPSD and PkPSD enzymes as well as native enzymes from C. albicans and mouse mitochondrial extracts.

FIGs. 7A-7D show efficacy of YU253467 and YU254403 against non-albicans Candida species. FIGs. 7A-7C show activity of YU253467 and YU254403 against C. albicans , C. glabrata , and C. parapsilosis at 75 or 200 p /mL in the absence or presence of 2mM ethanolamine. FIG. 7D shows in vitro efficacy of combinations of YU254403 with either fluconazole, amphotericin B or terbinafme. Compounds were evaluated at their MIC50 alone or in combination against C. albicans using the 48 h growth assay.

FIGs. 8A-8B show efficacy of YU254403 against mold pathogens. Activity of YU254403 against (FIG. 8 A) A. fumigatus and (FIG. 8B) Fusarium solani ( var .

petroliphiluni) at 9, 18, 36 and 72 pg/mL in the absence or presence of 2 mM ethanolamine.

BRIEF SUMMARY OF THE DISCLOSURE

In certain embodiments, compounds of Formula I are provided. In certain embodiments, the compound of Formula I, or a salt, solvate, tautomer, enantiomer, and/or diastereoisomer thereof, has the structure:

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, NO2, S(0₂)0R', C(0)0R', C(0)NR'R', and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, NO2, S(0₂)0R", C(0)0R", C(0)NR"R", and CN;

each occurrence of R'is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5.

Also provided are methods of screening a plurality of drug candidate compounds against a target phosphatidylserine decarboxylase (PSD) enzyme. In certain embodiments, the method comprises providing a PSD enzyme into each of a plurality of sample wells. In certain embodiments, the method comprises adding a candidate drug compound to at least one of the plurality of sample wells. In certain embodiments, the method comprises optically detecting an effect of the drug candidate compound on the PSD enzyme.

Also provided are methods of killing or disinfecting bacteria, yeast, or fungus. In certain embodiments, the method comprises contacting a bacterial, yeast, or fungal population with a compound of Formula I, wherein the bacterial, yeast, or fungal population is killed or disinfected after coming into contact with the compound of Formula I.

Advantageously, in some embodiments, the methods described herein can identify compounds that bind to PSD. Surprisingly, certain compounds identified with the methods herein can kill or disinfect bacteria, yeast, or fungus.

DETAILED DESCRIPTION OF THE DISCLOSURE

Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

Definitions

The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of' can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

The term "organic group" as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(0)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, S0₂R, S0₂N(R)₂, S0₃R, C(0)R, C(0)C(0)R,

C(0)CH₂C(0)R, C(S)R, C(0)OR, OC(0)R, C(0)N(R)_¾ OC(0)N(R)₂, C(S)N(R)₂, (CH₂)_O- ₂N(R)C(0)R, (CH₂)_O.2N(R)N(R)₂, N(R)N(R)C(0)R, N(R)N(R)C(0)OR, N(R)N(R)CON(R)₂, N(R)S0₂R, N(R)S0₂N(R)₂, N(R)C(0)OR, N(R)C(0)R, N(R)C(S)R, N(R)C(0)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(=NH)N(R)₂, C(0)N(OR)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

The term "substituted" as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen ( e.g F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(0)N(R)₂, CN, NO, NO₂, ONO2, azido, CF₃, OCF₃, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, S0₂R, S0₂N(R)₂, S0₃R, C(0)R, C(0)C(0)R,

C(0)CH₂C(0)R, C(S)R, C(0)OR, OC(0)R, C(0)N(R)₂, OC(0)N(R)₂, C(S)N(R)₂, (CH₂)_O. ₂N(R)C(0)R, (CH₂)_O.2N(R)N(R)₂, N(R)N(R)C(0)R, N(R)N(R)C(0)OR, N(R)N(R)CON(R)₂, N(R)S0₂R, N(R)S0₂N(R)₂, N(R)C(0)OR, N(R)C(0)R, N(R)C(S)R, N(R)C(0)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(=NH)N(R)₂, C(0)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (Ci- Cioo)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

The term "alkyl" as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.

Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.

Examples include, but are not limited to vinyl, -CH=C=CCH₂, -CH=CH(CH₃), - CH=C(CH₃)₂, -C(CH₃)=CH₂, -C(CH₃)=CH(CH₃), -C(CH₂CH₃)=CH₂, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

The term "alkynyl" as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - CºCH, -CºC(CH₃), -CºC(CH₂CH₃), -CH₂CºCH, -CH₂CºC(CH₃), and -CH₂CºC(CH₂CH₃) among others.

The term "acyl" as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkyl alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridyl acetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

The term "cycloalkyl" as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono- substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-sub stituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term

"cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

The term "aryl" as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

The term "aralkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

The term "heterocyclyl" as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl,

dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono- substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.

The term "heteroaryl" as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N,

O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.

Likewise a C₄-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein.

Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3 -(2, 3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),

6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1 -indolyl, 2-indolyl,

3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl,

4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl,

7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-l-yl, 5H-dibenz[b,f]azepine-2-yl,

5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl),

10,1 l-dihydro-5H-dibenz[b,f]azepine (10, 1 l-dihydro-5H-dibenz[b,f]azepine-l-yl,

10,1 l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-3-yl,

10,1 l-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3 -yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl

The term "heteroarylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

The term "alkoxy" as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a

methoxy ethoxy group is also an alkoxy group within the meaning herein, as is a

methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

The term "amine" as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH₂, for example, alkylamines, arylamines, alkylarylamines; R₂NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

The term "amino group" as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR₃ ⁺, wherein each R is independently selected, and protonated forms of each, except for - R₃ ⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

The terms "halo," "halogen," or "halide" group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

The term "haloalkyl" group, as used herein, includes mono-halo alkyl groups, poly halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.

The terms "epoxy-functional" or "epoxy-substituted" as used herein refers to a functional group in which an oxygen atom, the epoxy substituent, is directly attached to two adjacent carbon atoms of a carbon chain or ring system. Examples of epoxy-substituted functional groups include, but are not limited to, 2,3-epoxypropyl, 3,4-epoxybutyl, 4,5- epoxypentyl, 2,3-epoxypropoxy, epoxypropoxypropyl, 2-glycidoxyethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2~(glycidoxycarbony!)propyi, 3-(3,4-epoxycylohexyl)propyl, 2-(3 ,4- epoxy cyclohexyl jethyl, 2-(2,3-epoxycylopentyl)ethyl, 2-(4-methyl-3,4- epoxycyclohexyl)propyl, 2-(3,4-epoxy-3-methylcylohexyl)-2-methylethyl, and 5,6- epoxy hexyl.

The term "monovalent" as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

The term "hydrocarbon" or "hydrocarbyl" as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.

As used herein, the term "hydrocarbyl" refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (C_a- C’_b)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Ci), ethyl (C2), propyl (C3), or butyl (C4), and (Co-C_b)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

The term "solvent" as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X¹, X², and X³ are independently selected from noble gases" would include the scenario where, for example, X¹, X², and X³ are all the same, where X , X , and X are all different, where X and X are the same but X is different, and other analogous permutations.

The term "room temperature" as used herein refers to a temperature of about 15 °C to

28 °C.

The term "standard temperature and pressure" as used herein refers to 20 °C and 101 kPa.

As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term "efficacy" refers to the maximal effect (Emax) achieved within an assay.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).

Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b-hydroxybutyric, salicylic, galactaric and galacturonic acid.

Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be

"acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin; talc, excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar, buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a

pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in

Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,

PA), which is incorporated herein by reference.

The terms "patient," "subject," or "individual" are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

As used herein, the term "potency" refers to the dose needed to produce half the maximal response (ED50).

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound or compounds as described herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient ( e.g ., for diagnosis or ex vivo applications), who has a condition contemplated herein or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, or the symptoms of a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

Preparation of Compounds

Compounds of Formula I or otherwise described herein can be prepared by the general schemes described herein, using the synthetic method known by those skilled in the art The following examples illustrate non-limiting embodiments of the compound(s) described herein and their preparation.

In various embodiments, a compound of Formula I, or a salt, solvate, tautomer, enantiomer, and/or diastereoisomer thereof, has the structure:

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen, F, Cl, Br, I, NO2, S(0₂)0R', C(0)0R', C(0)NR'R', and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen, F, Cl, Br, I, N0₂, S(0₂)0R", C(0)0R", C(0)NR"R", and CN;

each occurrence of R'is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5.

In various embodiments, R¹ is hydrogen. In various embodiment, the compound has the structure:

In various embodiments, the compound has the structure:

In various embodiments, the compound of Formula I is not:

In various embodiments, the compound of Formula I is not:

The compounds described herein can possess one or more stereocenters, and each stereocenter can exist independently in either the ( R ) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms.

It is to be understood that the compounds described herein encompass racemic, optically- active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically -active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including

stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and / or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether ( e.g .,

tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.

In certain embodiments, the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein.

In certain embodiments, compounds described herein are prepared as prodrugs. A “prodrug“ refers to an agent that is converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

In certain embodiments, sites on, for example, the aromatic ring portion of compound(s) described herein are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.

Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ²H, ³H, ⁿC, ¹³C, ¹⁴C, ³⁶C1, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³²P, and ³⁵S.

In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as C, F, O and N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (lohn Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4^th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein.

In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In other embodiments, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal. In certain embodiments, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.

In certain embodiments, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively -removable protective groups such as 2,4-dimethoxybenzyl, while co existing amino groups are blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosure.

Compositions

The compositions containing the compound(s) described herein include a pharmaceutical composition comprising at least one compound as described herein and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal ( e.g sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Methods of Screening Drug Candidates

The role of phosphatidyl serine decarboxylases in cellular metabolism and pathogenesis is an emerging line of inquiry supported by compelling evidence that positions the PSDs as novel therapeutic targets in parasitic, fungal, and human neoplastic disease. A non-limiting method of screening a plurality of drug candidate compounds against a target phosphatidylserine decarboxylase (PSD) enzyme includes providing a PSD enzyme into each of a plurality of sample wells, adding a candidate drug compound to at least one of the plurality of sample wells; and optically detecting an effect of the drug candidate compound on the PSD enzyme. The current body of work indicates a target class with significant opportunity across multiple indications. Accordingly, the emerging understanding of the role for PSDs in microbial pathogenesis and cancer have stimulated efforts to identify selective inhibitors for use in validating the enzymes as drug targets, several of which are detailed herein. While the PSDs have attracted interest as drug targets based on the centrality of membrane phospholipid homeostasis to a variety of microbial pathogens as well as to the tumor microenvironment, they have also been described as undruggable.

In various embodiments, the plurality of sample wells comprise a reaction medium comprising phosphatidylserine. In various embodiments, the PSD enzyme is a Plasmodium knowlesi enzyme. In various embodiments, the method includes reacting the PSD enzyme with the drug candidate compound in the reaction medium.

In various embodiments, the reacting occurs at a temperature of about 22 °C to 28 °C for a period of about 50 minutes to 90 minutes. In various embodiments, the reacting occurs at a temperature of about 22, 23, 24, 25, 26, 27, or about 28 °C. In certain embodiments, the reacting occurs for a period of about 50, 55, 60, 65, 70, 75, 80, 85, or about 90 minutes. In certain embodiments, after the period for the reaction described herein, the pH of the reaction medium is set to about pH 8 to about pH 10. In certain embodiments, the pH of the reaction medium is set to about pH 8, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, or about 10.

In some embodiments, the method of screening drug candidates further includes adding distyrylbenzene-bis-aldehyde to the reaction medium, and incubating the reaction medium in the dark for about 1 to 4 hours. In various embodiments, the distyrylbenzene-bis- aldehyde is 4,4'-((lif,TA)-(2,5-bis((2,5,8, 12,15, 18-hexaoxanonadecan-10-yl)oxy)-l,4- pheny lene)bis(ethene-2, 1 -diyl))dibenzaldehy de (D SB -3 ) .

DSB-3 In various embodiments, the incubating occurs for about 1, 1.5, 2, 2.5, 3, 3.5, or about 4 hours. In various embodiments, the optically detecting an effect of the drug candidate compound on the PSD enzyme includes detecting fluorescence in at least one of the plurality of sample wells. In some embodiments, the fluorescence is quantified using light with X_cs of 403 nm and light emission is detected at a l _i1 of 508 nm.

Methods of Treatment and Use

A method of killing or disinfecting bacteria, yeast, or fungus includes, in various embodiments, contacting a bacterial, yeast, or fungal population with a compound of Formula I, wherein the bacterial, yeast, or fungal population is killed or disinfected after coming into contact with the compound of Formula I.

The compounds of Formula I have anti -bacterial and anti-fungal properties, and are able to kill at least, or greater than about 95%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% of bacteria, yeast, or fungus that come in contact with or are exposed to the compounds of Formula I. In various embodiments, the bacteria that are killed are pathogenic bacteria, pathogenic yeast, and/or pathogenic fungus that cause deleterious infections and/or diseases in mammals. In various embodiments, the bacteria are Gram-positive or Gram-negative. In various embodiments, the mammal is a cat, dog, human, sheep, horse, mouse, rabbit, rat, cow, goat, pig, and the like. The term "kill" as used herein means that the bacteria, yeast, or fungus is/are no longer able to exhibit or produce any harmful effect to or in a living organism, and/or that the bacteria, yeast, or fungus is/are unable to cause further infection, and/or the bacteria, yeast, or fungus cease to live.

The types of bacteria, yeast, or fungus that can be killed by the compounds of Formula I includes C. albicans, although other types of bacterial, yeast, or fungal organisms can be similarly killed. Non-limiting examples of bacteria genera that are killed when exposed to compounds of Formula I include Bacillus , Bartonella , Bordetella , Borrelia , Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus , Escherichia , Francisella , Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

In various embodiments, the pathogenic bacteria, yeast, or fungus can be C. albicans wild type (strain SC5314), C. albicans mutant (psdlD/psdlD and psd2D/psd2D), C. parapsilosis (strain ATCC 22019), C. glabrata (strain CSH10 CAG-1), A. fumigatus (strain CEA10) and Fusarium solani (var. petroliphilum).

In various embodiments, wild type C. albicans is sensitive to YU253467 and

YU254403 and in some embodiments is modulated by the availability of ethanolamine in the medium. For example, in some embodiments the YU253467 and YU254403 MIC50 values increased by 3.3 x in the case of YU253467 and by 4 x in the case of YU254403 in the presence of ethanolamine. Without being bound by theory, and because the primary cellular source of PE in the absence of exogenous supplementation is driven by PSD conversion of endogenous PS to PE, the sensitivity of these compounds to ethanolamine supplementation (which creates an effective shunt around PSD) is believed to occur PSD inhibition.

Interestingly, the inhibitors were not as effective at 75 pg/mL against the C. albicans psd2 \ \ mutant (FIG. 6A). While the exact mechanism for psd2A/A resistance to the compounds remains to be elucidated, without wishing to be limited by any theory, possible explanations include increased activity of the endogenous Psdl in the mutant to compensate for the loss of Psd2, or increased activity in other metabolic processes or detoxification mechanisms caused by the loss of Psd2. However, ethanolamine in the host is limiting for C. albicans. In addition, compensation mechanisms, such as potential enhancement of Psdl, can be overcome by increasing the drug concentration to 200 pg/mL (FIG. 6A).

In addition to the various species of Candida yeasts, the efficacy of YU254403 was tested against two mold (filamentous) pathogens: Aspergillus fumigatus and Fusarium solani ( var . petroliphilum). A. fumigatus is the predominant cause of invasive aspergillosis, which carries a mortality rates as high as 95% in immunocompromised patients. This underscores a critical need for novel antifungal modalities to combat this organism and, to this end, YU254403 demonstrated marked activity in both the presence and absence of ethanolamine. By contrast, a weak activity of YU254403 against l·^'. solani (var. petroliphilum) was observed, a significant cause of fungal keratitis.

The methods described herein include administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated in a pharmaceutical composition. In various embodiments, a therapeutically effective amount of at least one compound described herein present in a pharmaceutical composition is the only therapeutically active compound in a pharmaceutical composition. In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent that kills or disinfects bacteria.

In certain embodiments, administering the compound(s) described herein to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in killing or disinfecting bacteria in the subject For example, in certain embodiments, the compound(s) described herein enhance(s) the activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect.

In various embodiments, the compounds of Formula I can be used to disinfect non living objects, including non-living objects or surfaces made from metals, ceramics, glass, wood, fabrics, rubber, plastic, polymers, and composite materials made from any

combination of the foregoing, and combinations thereof. In various embodiments, the bacterial population is present on or in a non-living object. As used herein, the term

"disinfect" means at least about 95%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% of bacteria on or in a non living object that come into contact with or are exposed to the compounds of Formula I are no longer able to exhibit or produce any harmful effect to or in a living organism, and/or that the bacteria are unable to cause further infection, and/or the bacteria cease to live. In various embodiments, the bacteria disinfected on or from a non-living surface are pathogenic bacteria that can cause deleterious infections and/or diseases in mammals.

The compound of Formula I can also be formulated in a non-pharmaceutical composition that is intended for use on non-living objects or is used on living objects.

Suitable non-pharmaceutical compositions can be in the form of sprays, gels, slow-dissolving tablets ( e.g . for a toilet water reservoir), porous materials that incorporate the compounds of Formula I, and the like. The non-pharmaceutical compositions can include one or more excipients such as surfactants (cationic, anionic, neutral), emulsifiers, fragrances, thickening agents, artificial colors or dyes, detergents, water, salts, buffers, and the like.

In certain embodiments, the compound(s) described herein and the therapeutic agent are co-administered to the subject. In other embodiments, the compound(s) described herein and the therapeutic agent are coformulated and co-administered to the subject.

In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

Combination Therapies

The compounds useful within the methods described herein can be used in combination with one or more additional therapeutic agents useful for treating bacterial, yeast, or fungal infections. These additional therapeutic agents may comprise compounds that are commercially available or synthetically accessible to those skilled in the art. These additional therapeutic agents are known to treat or reduce the symptoms, of a bacterial, yeast, or fungal infection.

In various embodiments, a synergistic effect is observed when a compound as described herein is administered with one or more additional therapeutic agents or compounds. A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E_max equation (Holford & Scheiner, 1981, Clin.

Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a bacterial, yeast, or fungal infection. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be

continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions described herein to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a bacterial, yeast, or fungal infection in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a bacterial, yeast, or fungal infection in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of

compounding/formulating such a therapeutic compound.

In certain embodiments, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus,

administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account.

The compound(s) described herein for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 350 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, a composition as described herein is a packaged

pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second

pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents.

Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

For oral administration, the compound(s) described herein can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents ( e.g ., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OP ADR Y™ film coating systems available from Colorcon, West Point, Pa. (e.g., OP ADR Y™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and

OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g, lecithin or acacia); non-aqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).

Compositions as described herein can be prepared, packaged, or sold in a formulation suitable for oral or buccal administration. A tablet that includes a compound as described herein can, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, dispersing agents, surface-active agents, disintegrating agents, binding agents, and lubricating agents.

Suitable dispersing agents include, but are not limited to, potato starch, sodium starch glycollate, poloxamer 407, or poloxamer 188. One or more dispersing agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more dispersing agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Surface-active agents (surfactants) include cationic, anionic, or non-ionic surfactants, or combinations thereof. Suitable surfactants include, but are not limited to, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, carbethopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridine chloride, didecyldimethylammonium chloride,

dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, domiphen bromide, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, tetramethylammonium hydroxide, thonzonium bromide, stearalkonium chloride, octenidine dihydrochloride, olaflur, N-oleyl-l,3-propanediamine, 2-acrylamido-2-methylpropane sulfonic acid, alkylbenzene sulfonates, ammonium lauryl sulfate, ammonium perfluorononanoate, docusate, di sodium cocoamphodiacetate, magnesium laureth sulfate, perfluorobutanesulfonic acid,

perfluorononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate, sodium laurate, sodium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium

nonanoyloxybenzenesulfonate, sodium pareth sulfate, sodium stearate, sodium sulfosuccinate esters, cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide diethanolamine, cocamide monoethanolamine, decyl glucoside, decyl polyglucose, glycerol monostearate, octylphenoxypolyethoxy ethanol CA-630, isoceteth-20, lauryl glucoside, octylphenoxypolyethoxyethanol P-40, Nonoxynol-9, Nonoxynols, nonyl phenoxypolyethoxylethanol (NP-40), octaethylene glycol monododecyl ether, N-octyl beta- D-thioglucopyranoside, octyl glucoside, oleyl alcohol, PEG- 10 sunflower glycerides, pentaethylene glycol monododecyl ether, polidocanol, poloxamer, poloxamer 407, polyethoxylated tallow amine, polyglycerol polyricinoleate, polysorbate, polysorbate 20, polysorbate 80, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, and Tween 80. One or more surfactants can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more surfactants can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Suitable diluents include, but are not limited to, calcium carbonate, magnesium carbonate, magnesium oxide, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate, CELLACTOSE ® 80 (75 % a -lactose monohydrate and 25 % cellulose powder), mannitol, pre-gelatinized starch, starch, sucrose, sodium chloride, talc, anhydrous lactose, and granulated lactose. One or more diluents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more diluents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Suitable granulating and disintegrating agents include, but are not limited to, sucrose, copovidone, corn starch, microcrystalline cellulose, methyl cellulose, sodium starch glycollate, pregelatinized starch, povidone, sodium carboxy methyl cellulose, sodium alginate, citric acid, croscarmellose sodium, cellulose, carboxymethylcellulose calcium, colloidal silicone dioxide, crosspovidone and alginic acid. One or more granulating or disintegrating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more granulating or disintegrating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Suitable binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, anhydrous lactose, lactose monohydrate, hydroxypropyl methylcellulose, methylcellulose, povidone, polyacrylamides, sucrose, dextrose, maltose, gelatin, polyethylene glycol. One or more binding agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more binding agents can each be individually present in the

composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Suitable lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, hydrogenated castor oil, glyceryl monostearate, glyceryl behenate, mineral oil, polyethylene glycol, poloxamer 407, poloxamer 188, sodium laureth sulfate, sodium benzoate, stearic acid, sodium stearyl fumarate, silica, and talc. One or more lubricating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more lubricating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.

Tablets can be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U S. Patent Nos. 4,256, 108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation.

Tablets can also be enterically coated such that the coating begins to dissolve at a certain pH, such as at about pH 5.0 to about pH 7.5, thereby releasing a compound as described herein. The coating can contain, for example, EUDRAGIT ® L, S, FS, and/or E polymers with acidic or alkaline groups to allow release of a compound as described herein in a particular location, including in any desired section(s) of the intestine. The coating can also contain, for example, EUDRAGIT ® RL and/or RS polymers with cationic or neutral groups to allow for time controlled release of a compound as described hrein by pH-independent swelling.

Parenteral Administration

For parenteral administration, the compounds as described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.

Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural

pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.

Additional Administration Forms

Additional dosage forms suitable for use with the compound(s) and compositions described herein include dosage forms as described in U.S. Patents Nos. 6,340,475;

6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062;

20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757. Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations described herein can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use with the method(s) described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In some cases, the dosage forms to be used can be provided as slow or controlled- release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profde in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions described herein. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the compositions and dosage forms described herein.

Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects. Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being

metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient. In various embodiments, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. In various embodiments, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Dosing

The therapeutically effective amount or dose of a compound described herein depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a bacterial, yeast, or fungal infection in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.

The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on

Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the compound(s) described herein is optionally given continuously;

alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday“). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%,

40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.

The compounds described herein can be formulated in unit dosage form. The term “unit dosage form“ refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses ( e.g ., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED₅₀. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

Examples

Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

Experimental Procedures

Expression and purification of MBP-His6-A34PkPSD.

A Rosetta DE3 strain harboring a pMAL-C2X-His6-A34PkPSD was grown overnight in 10 mL of LB medium supplemented with 0.2% glucose, ampicillin (100 pg/mL) and chloramphenicol (34 pg/mL) at 37 °C. The following day, cells were inoculated into 1 liter of fresh medium and then grown to an Ar,oo -0.5. Expression of the MBP- His6-A34PkPSD was initiated by addition of 3 mL of 0.1 M isopropylthiogalactoside (IPTG). After incubation for 2 hours at 37 °C, the cells were harvested and washed by resuspension in water and re centrifugation. The cells were re-suspended in 25 mL of a column buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10 mM b mercaptoethanol (b-ME), stored overnight at -20 °C. The next day, the frozen cells were slowly thawed on ice water and then broken by soni cation (15 second burst at 30% amplitude using a Fisher Sonic Dismembrator 500, performed 8 times with 30 second on ice between intervals). Cell free extracts were obtained by centrifugation at 20,000 x g for 20 minutes at 4 °C. MBP-His6-A34PkPSD protein was purified by amylose affinity chromatography according to the manufacture’ s protocol (New England Biolabs #E8200S). The fractions containing the MBP-His6- A34PkPSD protein were identified by western blot analysis using anti-His6 antibody

(Clontech #631212).

PSD activity assay using ¹⁴C-PS as a substrate.

Purified MBP-His6-A34PkPSD, purified MBP-A40PfPSD, and mitochondria prepared from C. albicans strains and HeLa Ohio cells were used in a radiochemical PSD enzyme assay. The assay contained 50 mM ¹⁴C-PS (4,000 cpm/nmol) as the substrate (~K_m concentration for PkPSD) in the presence of specified concentrations of inhibitor molecules, and the reaction product was trapped as ¹⁴C0₂ on 2 M KOH-impregnated filter paper.

384-well HTS assay for PSD Inhibitors.

The HTS was performed using commercially available compound libraries or acquired compounds formatted as 10 mM stocks in dimethyl sulfoxide. The screening collection for this campaign was comprised of Screen-Well FDA-approved drugs (Enzo Life Sciences), Screen -Well Kinase inhibitors (Enzo Life Sciences), Microsource Pharmakon 1600 (MicroSource Discovery Systems), SelleckChem Kinase Inhibitors (Selleckchem), a “Tested in Humans” Collection (Yale Center for Molecular Discovery), and bioactive collections from ChemDiv, ChemBridge, and Analyticon. More information about the Bioactive Collections can be found at ycmd dot yale dot edu/smallmoleculecollections. Freshly prepared assay reagents and buffers were frozen at -80°C in convenient aliquots and used within a month of preparation. The thawed MBP- Hise-A34PkPSD was diluted in buffer A-l (100 mM NaCl, 16 pM EDTA, 160 mM b-mercaptoethanol, 319 mM Tris-HCl, 1 mM potassium phosphate (K₃PO₄) buffer, pH 7.4). The assay was conducted in black-wall, flat bottom, opaque, untreated 384 well microtiter plates (Corning 3575). The diluted enzyme preparation (10 pL/well) was dispensed using a small cassette Multidrop Combi dispenser (Thermo Fisher Scientific) for active enzyme and a multichannel pipette for heat-inactivated (negative control) enzyme. The plates were centrifuged (1000 RPM/30 s) to ensure reagent mixing. 20 nL aliquots of 10 mM library compounds were transferred to the wells via acoustic dispensing on the Echo 550 (LabCyte), and the plates were again centrifuged and subsequently incubated for 20 minutes at room temperature to allow binding of test compounds to the PSD enzyme. The PSD assay was initiated by addition of 10 pL of PS lipid substrate to appropriate wells using the small cassette combidrop, followed by centrifugation. Control reactions consisting of either detergent (no substrate control), or PE were dispensed with a multi-channel pipettor. Plates were then incubated at room

temperature for 75 minutes to allow the enzymatic conversion of PS to PE to proceed in the presence of test compounds. The final assay conditions were: 50 mM NaCl, 0.75 mM

TX100, 50 mM PS (or 50 mM PE control), 80 mM b-mercaptoethanol, 160 mM Tris-HCl, 1 mM K3PO4, pH 7.4, 30 ng MBP-His6-A34PkPSD (or boiled enzyme control) in a volume of 20 pL. The enzyme reaction was arrested by addition of 2.5 pL of 100 mM sodium tetraborate buffer (pH 9) and brief centrifugation. Subsequently, 2.5 pL of 100 pM DSB-3 in 1 mM KH2PO4, pH 7.4, was added to the plate, followed by brief centrifugation, and incubation for 2 hours in the dark. The PSD activity was monitored by measuring fluorescence intensities (403 ex/508 em, TECAN).

HTA data analysis and normalization.

Within plates, primary HTS data were normalized as follows: low signal (positive control, analogous to total PSD1 inhibition by a test compound) was 16 wells containing screening concentrations of PS + heat denatured PSD + DSB3. In this setting, no enzymatic interconversion of PS to PE occurred, thus the emission shift associated with binding of PS and DSB-3 was negligible. High signal (negative control, analogous to no inhibition of PSD by a test compound) was 16 wells containing screening concentrations of PS + active PSD + DSB3. Primary data were expressed as % of the positive control wells, and Z-prime and S B were calculated between the positive and negative controls. Secondary dose- response data were normalized as above. Normalized % inhibition data were fitted for IC50 determination via 4 parameter logistic equation. All offline data transformation and curve fitting was performed in ActivityBase (Abase) high content and throughput screening software (IDBS).

Strains.

C. albicans wild type (strain SC5314) and mutant (psdl D psdl D and r^ά2D psd2 ), C. parapsilosis (strain ATCC 22019), C. glabrata (strain CSH10 CAG-1), A. fumigatus (strain CEA10) and Fusarium solani ( var . petroliphilum, a fungal keratitis clinical isolate) were used in this study. Growth assays.

For liquid assay in 96-well plate format, C. albicans cells were pre-cultured overnight in liquid YPD medium at 30 °C, washed three times in water and diluted to 10³ cells per well in 100 pL of DOB medium in the absence or presence of 2 mM ethanolamine (pH 6.5). All plates were incubated at 30 °C. A_{W I} measurements were taken with a BioTek SynergyMx microplate reader every 8 hours for a total of 50 hrs. The compounds YU253467,

YU253454, YU224252, YU196325, YU254403 were tested at 0, 15, 30, and 60 pg/mL.

Fluconazole was used as a positive control and tested at concentrations 0, 3 and 10 ug/mL MIC50 values were calculated at 48 hr for YU253643 and fluconazole concentrations ranging from 5-100 pg/mL and 1-10 pg/mL, respectively.

Aspergillus fumigatus growth assays: Conidia were harvested from Sabouraud’s dextrose agar plates (Difco), fdtered through Miracloth (Millipore), washed twice in PBS, and inoculated to a density of 1.0 x lOVmL in glucose minimal medium (containing 10 mM ammonium tartrate) or RPMI-1640 (pH 7; supplemented with 1.8% glucose), with or without 2 mM ethanolamine (pH 6.5). 100 pL of sample were transferred into wells of 96-well plates and incubated at 35°C for 48h in the presence of 0, 9, 18, 36, 72 pg/mL of YU254403.

Absorbance was read at 530 nm in a CLARIOstar (BMG Labtech) plate reader.

Fusarmm solani (var. petroliphilum) growth assays: Microconidia were collected from yeast extract peptone (YPD) liquid cultures by fdtration through Miracloth and washed twice with PBS. Growth assays were otherwise performed as described above for A.

fumigatus in glucose minimal medium.

HeLa and HepG2 cell toxicity assays.

ATCC HeLa and HepG2 cells were dispensed into sterile black-walled, clear bottom, tissue culture treated, 384-well plates (Corning cat#3712) via MultiDrop (Thermo) at a density of 400 cells/well in a volume of 20 pL complete media. Cell plates were centrifuged at 46 g for 10 sec and incubated overnight at 37 °C in a humidified 5% CO2 incubator. 24 h after cell plating, test compounds (20 nL) were transferred from the compound source plate to the cell assay plate via Echo 550 acoustic dispenser (Labcyte). The final concentration of test compounds and DMSO were 10 pM and 0.1%, respectively. Tamoxifen (60 pM final assay concentration) was added to columns 1-2 as a positive control (maximum cell death), and columns 23-24 received DMSO vehicle only (negative control). Assay plates were centrifuged at 46 g for 10 sec and incubated for 72 h at 37 °C in a humidified 5% CO2 incubator. CellTiter-Glo (Promega) was used to measure cell viability in the assay wells according to the manufacturer’s instructions.

Briefly, CellTiter-Glo reagent (20 pL/well) was added to the assay plates using the MultiDrop dispenser. The plates were shaken on a Thermomixer R (Eppendorf) at 1,100 rpm for 1 min and incubated in the dark for 10 min at room temperature. Luminescence was measured in the Synergy Neo2 plate reader (BioTek) with 0.3 second sampling time per well. Wells displaying cytotoxicity have lower luminescence signals relative to the vehicle control wells. Raw data (luminescence counts per second) were normalized to Percent Effect by the formula 100 - (((sample - positive Ctrl mean) / (negative Ctrl mean - positive Ctrl mean))* 100).

High-throughput screening assay configuration.

The DSB3 -based biochemical assay can be run in both 96-well and 384-well format and is amenable to high-throughput screening. The optimized assay for HTS in 384-well plates was conducted in a 20 pL/wcll total reaction volume, with each test well containing 30 ng (-12.5 nM) A34PkPSD and 1 nanomole (50 mM) PS. The enzymatic reaction was performed at 24 °C for 75 minutes and terminated by shifting the reaction buffer pH to 9.0 with the addition of 10 mM sodium tetraborate. Subsequently, fluorescent adducts were generated by incubation with 10 mM DSB-3 in the dark for 2h (FIG. 1A). The fluorescence in each well was quantified using l_ec ⁼ 403 nm and X_em = 508 nm.

Primary HTS.

The primary HTS consisted of 130,858 compounds from several commercially- available bioactive and synthetic collections, screened at 10 mM final concentration in the DSB-3 biochemical assay with a single compound per well, and a single determination per compound. The primary screen was run in 12 assay iterations of 30-40 assay plates per run.

Each screening plate contained 16 replicates of the positive-inhibition control (PS + heat- denatured PSD+ DSB3), and 16 replicates of the negative-inhibition control (PE + active PSD + DSB3). To monitor assay performance, mean and standard deviations from control wells were used to quantify signal-to-background ratio (S/B) and Z-prime factor (Z’) for each screening plate. The distribution of S/B and Z’ scores across all plates is shown in FIGs. IB and 1C. Average Z’ was 0.78 ± 0.04, and average S/B, calculated as (PSD + PS + vehicle + DSB3) / (heat-inactivated PSD + PS + vehicle + DSB3), was 3.26 ± 0.12, confirming robustness of the high-throughput assay.

Raw screening data for library compounds was normalized relative to the mean of positive control wells (set as 100% inhibition) and negative control wells (set as 0% effect). FIG. ID depicts the normalized HTS data in scatter-plot format. Employing a mean % inhibition + 3SD cutoff for primary hit selection (20.6 ± 3.1%), the primary hit rate of 0.6% yielded 851 compounds for cherry-picking and subsequent retesting in triplicate from DMSO stocks.

Hit-picking and retests.

851 cherry-picked HTS hits were re-tested in triplicate at 10 mM using the primary HTS assay format (FIG. 2A). A parallel fluorescence interference counter-assay was employed to rule out non-specific compound interference with assay readout. In the interference assay, all wells received phosphatidylethanolamine and DSB-3 to generate maximal emission, and compounds were evaluated for ability to trigger false positives by quenching fluorescent emission in the absence of an enzymatic reaction. As depicted in FIG. 2B, this round of PSD retests and parallel interference assay identified 36 confirmed, non- fluorescent hits.

PSD concentration response and cytotoxicity counter-assays.

Confirmed HTS hits were advanced to biochemical concentration response studies from freshly solubilized dry powders to confirm the activity of the parent structure. Among the 36 confirmed hits, 15 were available commercially as dry powders (FIG. 3 A, filled circles). Compounds were dosed out in duplicate from a top concentration of 80 mM with normalization and curve fitting as described in Materials and Methods. After the initial concentration-response study of these 15 confirmed hits, 46 analogs were procured via custom synthesis, or via virtual hit expansion from the commercially available chemical space (FIG. 3 A, open circles). Lead candidates, which display PSD ICA, <40 mM, were selected for progression to cell-based anti-fungal assays. Of these compounds, three showed negligible fluorescence interference in the range of their in vitro IC50S against PKPSD. One compound, YU253467, showed fluorescence interference equivalent to its IC₅₀ against PkPSD; however, an orthogonal assay that measures production of ¹⁴C-PE from ¹⁴C-PS showed activity against PkPSD in vitro. Lead candidates were triaged versus potential toxicity to host cells via ATP quantification in live cells using a 72 h Cell-Titer Glo-based readout in HeLa cells. Potential for liver-specific cytotoxicity was evaluated by an identical parallel readout in HepG2 cells (cytotoxicity data not shown). Based on these triage criteria, four PSD-inhibiting small molecules displaying appreciable margins versus one or both cytotoxicity readouts progressed to cell- based anti-fungal assay testing (FIG. 3B).

Cell-based antifungal assay testing of confirmed HTS hits.

To evaluate the activity of YU253467, YU253454, YU224252 and YU196325 against C. albicans , cells were diluted to 10³ cells per well and incubated in minimal medium lacking or supplemented with ethanolamine, in the absence or presence of test compounds at concentrations of 15, 30 and 60 pg/mL. Of the 4 compounds tested, YU253467 and

YU253454 showed the highest inhibition of C. albicans growth in vitro (FIGs. 4A-4B). Notably, YU253467 showed strong activity at 15, 30 and 60 pg/mL in the absence of ethanolamine, with significant growth delay at 15 pg/mL, and 100% growth inhibition at 30 and 60 pg/mL. Ethanolamine supplementation rescued growth of C. albicans at 30 and 60 pg/mL, indicating that the compound mediated growth inhibition is on- mechanism at PSD.

In the control arm, fluconazole inhibition at 3 or 10 pg/mL was not affected by the presence or absence of ethanolamine (FIG. 4B).

Because YU253467 contains a chemotype well recognized as a pan-assay interference compound (PAINS), it was reasoned that reduction of the compound by removing the double bond would prevent the cyclo-reversion of this compound into its constituents, and thereby remove any interference from these breakdown products. The resulting compound,

YU254403, was subsequently synthesized, and its enzymatic inhibition and antifungal activity compared to that of the parent compound YU253467 (FIG. 5A).

Dose response assays showed MIC₅₀ against C. albicans of 22.5 pg/mL for

YU253467 and 15 pg/mL for YU254403 in the absence of ethanolamine (FIGs. 5B and 5C). As expected for PSD-specific inhibitors, the MIC50 of YU253467 increased from 22.5 pg/mL in the absence of ethanolamine to 75 pg/mL in the presence of ethanolamine, whereas that of YU254403 increased from 15 pg/mL in the absence of ethanolamine to 60 pg/mL in the presence of ethanolamine (FIGs. 5B and 5C). As a control, fluconazole displayed an MIC50 of 2.5 pg/mL, and a characteristic insensitivity to ethanolamine supplementation (FIG. 5D).

To further evaluate the link between PSD function and anti-C. albicans activity of YU253467 and YU254403, the effect of these compounds against psd 1 Apsd 1 A and psd2A^/psd2A C. albicans mutants lacking PSD1 or PSD2 genes was examined. Psdlp accounts for the majority of cellular PSD activity, as it’s loss results in ethanolamine auxotrophy. In the presence of ethanolamine, however, the inhibition profile of the psdl A/psdl A mutant was similar to wild type under similar conditions (FIG. 6A).

Interestingly, whereas the growth of the wild type strain was completely inhibited at 75 pg/mL and 200 pg/mL of YU253467 or YU254403 in the absence of ethanolamine, that of the psd2A/psd2A was not affected by these drugs at 75 pg/mL under similar conditions (FIG. 6A). Only at 200 pg/mL did the growth of the mutant resemble that of the wild type (FIG. 6A). However, in the presence of ethanolamine, the growth of the psd2A/psd2A strain was similar to that of the wild type at both 75pg/mL and 200 pg/mL of YU253467 or YU254403 (FIG. 6A).

Consistent with these genetic data, YU253467 and YU254403 at 72 pg/mL inhibited native C. albicans PSD activity in mitochondrial extracts by 94 and 63%, respectively (FIG. 6B). At this concentration, YU253467 inhibited the native mouse mitochondrial PSD activity by 41%, whereas YU254403 had little to no inhibitory activity. Since YU253467 was selected from a chemical screen using purified malarial PSD enzyme as a surrogate enzyme, activity assays of YU253467 and YU254403 at 72 pg/mL showed at least about 94 and at least about 96% inhibition of PfPSD, and at least about 92 and at least about 71% inhibition of PkPSD, respectively (FIG. 6B).

In vitro activity of YU253467 and YU254403 against other fungal pathogens.

The finding that YU253467 and YU254403 inhibit the growth of C. albicans led to investigate their activity against other Candida species. At 200 pg/mL, YU253467 and YU254403 at 200 pg/mL inhibited growth by at least about 70% and at least about 91% in the absence of ethanolamine, respectively, and by at least about 75% and at least about 55% in the presence of ethanolamine, respectively. In C. parapsilosis , 200 pg/mL of YU253467 and YU254403 results in at least about 57% and at least about 44% growth inhibition in the absence of ethanolamine and at least about 58% and at least about 46% inhibition in the presence of ethanolamine, respectively (FIGs. 7A-C). In vitro pairwise drug combination assays with C. albicans using YU254403 and one of three known antifungal drugs fluconazole, amphotericin B, and terbinafme did not show any synergistic or antagonistic effects (FIG. 7D). The range of activity of YU254403 was further examined against the mold pathogens Aspergillus fumigatus and Fusarium solani (var. petroliphilum) (FIG. 8). While no significant inhibition of Fusarium solani (var. petroliphilum) could be detected with these compounds (FIG. 8B), the growth of A. fumigatus was strongly inhibited by YU254403 with at least about 70% and at least about 40% inhibition at 18 and 36 pg/mL, respectively, in the absence of ethanolamine and at least about 45% and at least about 35% inhibition at 18 and 36 pg/mL, respectively, in the presence of ethanolamine (FIG. 8 A).

The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Enumerated Embodiments

The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

Embodiment 1 provides a method of screening a plurality of drug candidate compounds against a target phosphatidylserine decarboxylase (PSD) enzyme, the method comprising: providing a PSD enzyme into each of a plurality of sample wells; adding a candidate drug compound to at least one of the plurality of sample wells; and optically detecting an effect of the drug candidate compound on the PSD enzyme.

Embodiment 2 provides method of embodiment 1, wherein the plurality of sample wells comprise a reaction medium comprising phosphatidylserine.

Embodiment 3 provides the method of any one of embodiments 1-2, wherein the PSD enzyme is a Plasmodium knowlesi enzyme.

Embodiment 4 provides the method of any one of embodiments 1-3, comprising reacting the PSD enzyme with the drug candidate compound in the reaction medium.

Embodiment 5 provides the method of any one of embodiments 1-4, wherein the reacting occurs at a temperature of about 22 °C to 28 °C for a period of about 50 minutes to 90 minutes. Embodiment 6 provides the method of any one of embodiments 1-5, wherein after the period the pH of the reaction medium is set to about pH 8 to about pH 10.

Embodiment 7 provides the method of any one of embodiments 1-6, further comprising adding a distyrylbenzene-bis-aldehyde (DSB-3) to the reaction medium; and incubating the reaction medium in the dark for about 1 to 4 hours.

Embodiment 8 provides the method of any one of embodiments 1-7, wherein the optically detecting comprises detecting fluorescence in at least one of the plurality of sample wells.

Embodiment 9 provides the method of any one of embodiments 1-8, wherein the fluorescence is quantified using light with l_ec of 403 nm and wherein light emission is detected at a >__cm of 508 nm.

Embodiment 10 provides a compound of Formula I, or a salt, solvate, tautomer, enantiomer, and/or diastereoisomer thereof:

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen, F, Cl, Br, I, N0₂, S(0₂)OR', C(0)OR', C(0)NR'R, and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen, F, Cl, Br, I, N0₂, S(0₂)OR", C(0)OR", C(0)NR"R", and CN;

each occurrence of Ris independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5.

Embodiment 11 provides the compound embodiment 10, wherein R¹ is hydrogen. Embodiment 12 provides the compound of any one of embodiments 10-11, wherein the compound has the structure:

Embodiment 13 provides the compound of any one of embodiments 10-12, wherein the compound has the structure:

Embodiment 14 provides the compound of any one of embodiments 10-13, wherein the compound has the structure:

Embodiment 15 provides a method of killing or disinfecting bacteria, yeast, or fungus, the method comprising: contacting a bacterial, yeast, or fungal population with a compound of Formula I:

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, N0₂, S(0₂)OR, C(0)OR, C(0)NRR', and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, N0₂, S(0₂)0R", C(0)0R", C(0)NR"R", and CN;

each occurrence of R'is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5; wherein the bacterial, yeast, or fungal population is killed or disinfected after coming into contact with the compound.

Embodiment 16 provides the method of embodiment 15, wherein the bacterial, yeast, or fungal population comprises pathogenic bacteria, yeast, or fungus.

Embodiment 17 provides the method of any one of embodiments 15-16, wherein the pathogenic bacteria, yeast, or fungus comprises C. albicans wild type (strain SC5314), C. albicans mutant (psdlD/psdlD and psd2D/psd2D), C. parapsilosis (strain ATCC 22019), C. glabrata (strain CSH10 CAG-1), A.fumigatus (strain CEA10) and Fusarium solani ( var . petroliphilum) .

Embodiment 18 provides the method of any one of embodiments 15-17, wherein R¹ is hydrogen.

Embodiment 19 provides the method of any one of embodiments 15-18, wherein the compound has the structure:

Embodiment 20 provides the method of any one of embodiments 15-19, wherein the compound has the structure:

Embodiment 21 provides the method of any one of embodiments 15-20, wherein the compound has the structure:

Claims

CLAIMS What is claimed is:

1. A method of screening a plurality of drug candidate compounds against a target phosphatidylserine decarboxylase (PSD) enzyme, the method comprising:

providing a PSD enzyme into each of a plurality of sample wells;

adding a candidate drug compound to at least one of the plurality of sample wells; and optically detecting an effect of the drug candidate compound on the PSD enzyme.

2. The method of claim 1, wherein the plurality of sample wells comprise a reaction medium comprising phosphatidylserine.

3. The method of claim 1, wherein the PSD enzyme is a Plasmodium knowlesi enzyme.

4. The method of claim 2, comprising reacting the PSD enzyme with the drug candidate compound in the reaction medium.

5. The method of claim 4, wherein the reacting occurs at a temperature of about 22 °C to 28 °C for a period of about 50 minutes to 90 minutes.

6. The method of claim 5, wherein after the period the pH of the reaction medium is set to about pH 8 to about pH 10.

7. The method of claim 6, further comprising

adding a distyrylbenzene-bis-aldehyde (DSB-3) to the reaction medium; and incubating the reaction medium in the dark for about 1 to 4 hours.

8. The method of claim 7, wherein the optically detecting comprises detecting fluorescence in at least one of the plurality of sample wells.

9. The method of claim 8, wherein the fluorescence is quantified using light with l_ec of 403 nm and wherein light emission is detected at a _cm of 508 nm.

10. A compound of Formula I, or a salt, solvate, tautomer, enantiomer, and/or diastereoisomer thereof:

Formula I,

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen, F, Cl, Br, I, NO2, S(0₂)0R', C(0)0R', C(0)NR'R', and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen, F, Cl, Br, I, N0₂, S(0₂)0R", C(0)0R", C(0)NR"R", and CN;

each occurrence of Ris independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5.

11. The compound of claim 10, wherein R¹ is hydrogen.

12 The compound of claim 10, wherein the compound has the structure:

13. The compound of claim 10, wherein the compound has the structure:

14. The compound of claim 10, wherein the compound has the structure:

15. A method of killing or disinfecting bacteria, yeast, or fungus, the method comprising: contacting a bacterial, yeast, or fungal population with a compound of Formula I:

Formula I,

wherein

each occurrence of A¹ is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, N0₂, S(0₂)0R', C(0)0R', C(0)NR'R', and CN;

each occurrence of A² is independently selected from the group consisting of hydrogen,

F, Cl, Br, I, N0₂, S(0₂)0R", C(0)0R", C(0) R"R", and CN;

each occurrence of Ris independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; each occurrence of R is independently hydrogen, methyl, ethyl, n-propyl, or i-propyl; nl is 0, 1, 2, 3, 4, or 5;

n2 is 0, 1, 2, 3, 4, or 5;

wherein the bacterial, yeast, or fungal population is killed or disinfected after coming into contact with the compound.

16. The method of claim 15, wherein the bacterial, yeast, or fungal population comprises pathogenic bacteria, yeast, or fungus.

17. The method of claim 16, wherein the pathogenic bacteria, yeast, or fungus comprises C. albicans wild type (strain SC5314), C. albicans mutant (psdlD/psdlD and psd2D/psd2D), C. parapsilosis (strain ATCC 22019), C. glabrata (strain CSH10 CAG-1), A. fumigatus (strain CEA10) and Fusarium solani (yar. petroliphilum).

18. The method of claim 15, wherein R¹ is hydrogen.

19. The method of claim 15, wherein the compound has the structure:

20. The method of claim 15, wherein the compound has the structure:

21. The method of claim 15, wherein the compound has the structure: